The main objective of the proposed research is to study the first step of the excision-repair mechanism in normal human cells and in Fanconi's anemia (FA) fibroblasts. This step would be the recognition by an enzymatic activity(ies) (BPase) of the lesion(s) on DNA caused by the environmental carcinogen benzo(a)pyrene (B(a)P). The first experiments will be directed towards elucidation of the nature of the putative BPase, i.e, whether it is N-glycosylase and/or endonuclease. Substrates for BPase will include cellular, PM2 and calf thymus DNA modified by either unlabelled or radioactive benzo(a)pyrene diol epoxide I (BPDE) or by B(a)P, metabolized either by cells or by a rat liver microsomal activating system. BPase activity will be assayed in human placenta and in HeLa cells, and its nature characterized by the identification of the BPDE adducts removed from DNA. The adducts will be separated by chromatography on Sephadex LH20 and by reverse phase high pressure liquid chromatography (HPLC). Once the nature of BPase is known, its presence and activity will be evaluated in repair defective cell lines such as FA fibroblasts. To determine whether BPDE or B(a)P may actually suppress repair enzyme activity, BPase as well as apurinic endonuclease, uracil N-glycosylase and N-3-methyl adenine N-glycosylase activities will be assayed in both normal and FA cells following exposure to the carcinogens. These latter three enzymes, in purified form, will be tested in vitro against BPDE-modified DNA to determine whether they are active against any or all of the BPDE-induced lesions.